APPLICANT'S ABSTRACT: The long term Goals of this research are to understand the mechanisms which regulate mammalian alcohol dehydrogenase (ADH) gene expression, and the physiological and pathological consequences of alterations in ADH expression. It is our hypothesis that differences in the expression of the ADH genes can, like differences in ADH isozymes, affect the metabolism of alcohols and the consequences of alcohol consumption. A greater understanding of the regulation of the expression of the ADH genes will add much to our understanding of alcohol metabolism. This renewal continues our exploration of the cis-acting DNA sequences that are important in regulating ADH gene expression, and expands our study to the isolation and examination of the transcription factors that bind to these sites. We will identify cis-acting elements that are important in the regulation of tissue-specific expression of the human and mouse class I ADH genes ADH1, ADH2, ADH3 and Adh1. These elements will be studied by a combination of protein-binding analyses (such as DNaseI footprinting and gel retardation) and functional studies (transient transfections with reporter constructs). Comparisons among the closely related class I genes will reveal the effects of subtle chances in the cis-acting sequences, and will illuminate the evolution of tissue specificity. The ADH4, ADH5, ADH6 and ADH7 genes have different patterns of expression, and produce enzymes (PI-ADH, X-ADH, ADH6 and SIGMA-ADH, respectively) that influence important metabolic processes, including metabolism of many other alcohols such as retinol. We will examine the regulation of expression of these genes in parallel with our studies of the class I genes. We will attempt to determine which transcription factors bind to the cis- acting elements we identify, both by examining already described factors and by searching for novel factors. A major thrust of our efforts will be to clone transcription factors that bind to the cis-acting elements. We will screen cDNA expression libraries with oligonucleotides containing a cis- acting element. Since several of the cis-acting elements appear novel, we expect to clone novel transcription factors. Transcription factors will be analyzed to determine their binding specificity, and to identify functional domains within the protein. These studies will contribute both to our basic understanding of gene regulation, tissue specificity and development, and to our understanding of the genetic factors underlying differences among individuals in the metabolic, pharmacological and pathological effects of alcohol consumption.